Differentiation of human embryonic stem cells into pancreatic endocrine cells

ABSTRACT

The present invention provides methods to promote differentiation of pancreatic endoderm cells to pancreatic endocrine rich clusters and to enhance insulin expression in hormone-expressing cells.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. Ser. No. 13/911,829,filed Jun. 6, 2013 (now allowed), which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/657,160, filed Jun. 8, 2012,both of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention is in the field of cell differentiation. Morespecifically, the invention discloses use of Ephrin ligands andsphingosine-1-phosphate as regulators of differentiation of pluripotentstem cells to endocrine cells.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or β cells, appropriatefor engraftment. One approach is the generation of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, thyroid,thymus, pancreas, gut, and liver, will develop from the endoderm, via anintermediate stage. The intermediate stage in this process is theformation of definitive endoderm. Definitive endoderm cells express anumber of markers, such as, HNF3beta, GATA4, MIXL1, CXCR4 and SOX17.

By the end of gastrulation, the endoderm is partitioned intoanterior-posterior domains that can be recognized by the expression of apanel of factors that uniquely mark anterior, mid, and posterior regionsof the endoderm. For example, Hhex, and Sox2 identify the anteriorregion while Cdx1, 2, and 4 identify the posterior half of the endoderm.

Migration of endoderm tissue brings the endoderm into close proximitywith different mesodermal tissues that help in regionalization of thegut tube. This is accomplished by a plethora of secreted factors, suchas FGFs, Wnts, TGF-Bs, retinoic acid (RA), and BMP ligands and theirantagonists. For example, FGF4 and BMP promote Cdx2 expression in thepresumptive hindgut endoderm and repress expression of the anteriorgenes Hhex and SOX2 (2000 Development, 127:1563-1567). WNT signaling hasalso been shown to work in parallel to FGF signaling to promote hindgutdevelopment and inhibit foregut fate (2007 Development, 134:2207-2217).Lastly, secreted retinoic acid by mesenchyme regulates theforegut-hindgut boundary (2002 Curr Biol, 12:1215-1220).

The level of expression of specific transcription factors may be used todesignate the identity of a tissue. During transformation of thedefinitive endoderm into a primitive gut tube, the gut tube becomesregionalized into broad domains that can be observed at the molecularlevel by restricted gene expression patterns. For example, theregionalized pancreas domain in the gut tube shows a very highexpression of PDX1 and very low expression of CDX2 and SOX2. Similarly,the presence of high levels of Foxel are indicative of esophagus tissue;highly expressed in the lung tissue is NKX2.1; SOX2/Odd1 (OSR1) arehighly expressed in stomach tissue; expression of PROX1Hhex/AFP is highin liver tissue; SOX17 is highly expressed in biliary structure tissues;PDX1, NKX6.1/PTf1a, and NKX2.2 are highly expressed in pancreatictissue; and expression of CDX2 is high in intestine tissue. The summaryabove is adapted from Dev Dyn 2009, 238:29-42 and Annu Rev Cell Dev Biol2009, 25:221-251.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm (2009 Annu Rev Cell Dev Biol,25:221-251; 2009 Dev Dyn, 238:29-42). Dorsal and ventral pancreaticdomains arise from the foregut epithelium. Foregut also gives rise tothe esophagus, trachea, lungs, thyroid, stomach, liver, pancreas, andbile duct system.

Cells of the pancreatic endoderm express the pancreatic-duodenalhomeobox gene PDX1. In the absence of PDX1, the pancreas fails todevelop beyond the formation of ventral and dorsal buds. Thus, PDX1expression marks a critical step in pancreatic organogenesis. The maturepancreas contains, among other cell types, exocrine tissue and endocrinetissue. Exocrine and endocrine tissues arise from the differentiation ofpancreatic endoderm.

D'Amour et al. describes the production of enriched cultures of humanembryonic stem (ES) cell-derived definitive endoderm in the presence ofa high concentration of activin and low serum (Nature Biotechnol 2005,23:1534—1541; U.S. Pat. No. 7,704,738). Transplanting these cells underthe kidney capsule of mice resulted in differentiation into more maturecells with characteristics of endodermal tissue (U.S. Pat. No.7,704,738). Human embryonic stem cell-derived definitive endoderm cellscan be further differentiated into PDX1 positive cells after addition ofFGF-10 and retinoic acid (U.S. Patent Publication No. 2005/0266554A1).Subsequent transplantation of these pancreatic precursor cells in thefat pad of immune deficient mice resulted in formation of functionalpancreatic endocrine cells following a 3-4 month maturation phase (U.S.Pat. No. 7,993,920 and U.S. Pat. No. 7,534,608).

Fisk et al. report a system for producing pancreatic islet cells fromhuman embryonic stem cells (U.S. Pat. No. 7,033,831). In this case, thedifferentiation pathway was divided into three stages. Human embryonicstem cells were first differentiated to endoderm using a combination ofsodium butyrate and activin A (U.S. Pat. No. 7,326,572). The cells werethen cultured with BMP antagonists, such as Noggin, in combination withEGF or betacellulin to generate PDX1 positive cells. The terminaldifferentiation was induced by nicotinamide.

Small molecule inhibitors have also been used for induction ofpancreatic endocrine precursor cells. For example, small moleculeinhibitors of TGF-B receptor and BMP receptors (Development 2011,138:861-871; Diabetes 2011, 60:239-247) have been used to significantlyenhance number of pancreatic endocrine cells. In addition, smallmolecule activators have also been used to generate definitive endodermcells or pancreatic precursor cells (Curr Opin Cell Biol 2009,21:727-732; Nature Chem Biol 2009, 5:258-265).

Although great strides have been made in improving protocols to generatepancreatic cells from human pluripotent stem cells, there is still aneed to generate a protocol that results in functional endocrine cellsand in particular beta cells. Here, we demonstrate that a class ofEphrin ligands and sphingosine-1-phosphate or agonists of sphingosinereceptor enhance production of endocrine cells and accelerate clusteringof endocrine hormones and endocrine precursor cells.

SUMMARY

In an embodiment, the present invention relates to a method of enhancingexpression of insulin and NKX6.1 by culturing a population of pancreaticendoderm cells in medium comprising Ephrin A4 or Ephrin A3. In someembodiments, the population of pancreatic endoderm cells do notsubstantially express CDX2 or SOX2. In some embodiments, the populationpancreatic endoderm cells are obtained by a stepwise differentiation ofpluripotent cells. In some embodiments, the pluripotent cells are humanembryonic pluripotent cells.

In an embodiment, the invention concerns a method of enhancingexpression of somatostatin while suppressing the expression of insulin,glucagon, and ghrelin by culturing pancreatic endoderm cells in mediumcomprising Activin A or Activin C. In some embodiments, the populationof pancreatic endoderm cells treated with Activin A or Activin Cexpresses more somatostatin as a population of pancreatic endoderm cellsnon-treated with Activin A or Activin C. In some embodiments, theexpression of insulin is suppressed in the population of pancreaticendoderm cells treated with Activin A or Activin C as compared to theexpression of insulin in a population of pancreatic endoderm cellsnon-treated with Activin A or Activin C. In some embodiments, theexpression of glucagon in the population of pancreatic endoderm cellstreated with Activin A or Activin C is suppressed as compared to theexpression of glucagon in a population of pancreatic endoderm cellsnon-treated with Activin A or Activin C. In some embodiments, theexpression of ghrelin is suppressed in the population of pancreaticendoderm cells treated with Activin A or Activin C as compared to theexpression of ghrelin in a population of pancreatic endoderm cellsnon-treated with Activin A or Activin C . In some embodiments, thepancreatic endoderm cells do not substantially express CDX2 or SOX2. Insome embodiments, the pancreatic endoderm cells treated with Activin Aor Activin C are obtained by a stepwise differentiation of pluripotentcells. In some embodiments, the pluripotent cells where the pancreaticendoderm cells are derived from are human embryonic pluripotent cells.

In an embodiment, the invention refers to a method of enhancingexpression of NKX6.1 by treating pancreatic endoderm cells in mediumcomprising semaphorin 3a or Epigen. In some embodiments, the populationof pancreatic endoderm cells treated with medium comprising semaphorin3a or Epigen expresses an enhanced amount of NKX6.1 as compared topancreatic endoderm cells non-treated with medium comprising semaphorin3a or Epigen. In some embodiments, the level of expression of hormonessuch as insulin, glucagon, and gherlin is not affected in pancreaticendoderm cells treated with medium comprising semaphorin 3a or Epigen ascompared to pancreatic endoderm cells not treated with medium comprisingsemaphorin 3a or Epigen. In some embodiments, the pancreatic endodermcells do not substantially express CDX2 or SOX2. In some embodiments,the pancreatic endoderm cells treated with medium comprising semaphorin3a or Epigen are obtained by a stepwise differentiation of pluripotentcells. In some embodiments, the pluripotent cells where the pancreaticendoderm cells are derived from are human embryonic pluripotent cells.

In some embodiments, the present invention relates to a stepwise methodof differentiating pluripotent cells comprising culturing pancreaticendoderm cells in medium comprising Ephrin A4, Ephrin A3, Activin A,Activin C, semaphorin 3a, or Epigen. In some embodiments, the pancreaticendoderm cells are cultured in medium comprising Ephrin A4 or Ephrin A3.In some embodiments, the pancreatic endoderm cells are cultured inmedium comprising Activin A or Activin C. In some embodiments, thepancreatic endoderm cells are cultured in medium comprising semaphorin3a, or Epigen. In some embodiments, the pluripotent stem cells where thepancreatic endoderm cells are derived from are human embryonicpluripotent stem cells.

In an embodiment, the present invention relates to a method of inducingexpression of endocrine clusters by treating pancreatic endocrine cellswith sphingosine-1 receptor agonist. In some embodiments, thesphingosine-1 receptor agonist used for treating pancreatic endocrinecells is sphingosine-1-phosphate (S1P)

Also contemplated as embodiments of the invention are cells prepared bythe methods of the invention, and methods of using the cells of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1G shows data from real-time PCR analyses of theexpression of the following genes in cells of the human embryonic stemcell line H1 differentiated as described in Example 1: insulin (FIG.1A), somatostatin (FIG. 1B), ghrelin (FIG. 1C), glucagon (FIG. 1D), PDX1(FIG. 1E), NKX6.1 (FIG. 1F), and NGN3 (FIG. 1G).

FIG. 2A to FIG. 2C show images of cells immune stained for insulin. FIG.2A, control; FIG. 2B, cells treated with 50 ng/ml Ephrin-A3; and FIG.2C, cells treated with 100 ng/ml Ephrin-A3, as described in Example 2.

FIG. 3A to FIG. 3C show images of cells immune stained for insulin. FIG.3A, control; FIG. 3B, cells treated with 50 ng/ml Ephrin-A4; and FIG.3C, cells treated with 100 ng/ml Ephrin-A4, as described in Example 2.

FIG. 4A to FIG. 4D depict phase contrast images of S6 cultures of cellstreated with sphingosine-1-phosphate (S1P) and imaged on day 1 (FIG.4A), day 7 (FIG. 4B), and two different magnifications at day 10 (FIG.4C and FIG. 4D). The images show that on day 7, there was clear evidenceof clustering of endocrine cells and on day 10 the clusters wereseparated from each other by a thin layer of pancreatic endodermepithelium.

FIG. 5A to FIG. 5D depict images of cells treated with S1P andimmunostained for Hb9 (FIG. 5A) and NKX6.1 (FIG. 5B), or immunostainedfor insulin (FIG. 5C) and Hb9 (FIG. 5D).

FIG. 6A and FIG. 6B depict phase contrast images, at differentmagnifications, of cells treated with 10 μM S1P and harvested three daysafter start of stage 6. FIG. 6C and FIG. 6D depict images of cellsimmunostained for NKX2.2. FIG. 6C, control cells; FIG. 6D, cells treatedwith S1P.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability, at thesingle cell level, to both self-renew and differentiate. Stem cells mayproduce progeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm). Stem cells also give rise to tissues of multiplegerm layers following transplantation and contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors, and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiatedcell or a differentiation-induced cell is one that has taken on a morespecialized (“committed”) position within the lineage of a cell. Theterm “committed”, when applied to the process of differentiation, refersto a cell that has proceeded in the differentiation pathway to a pointwhere, under normal circumstances, it will continue to differentiateinto a specific cell type or subset of cell types, and cannot, undernormal circumstances, differentiate into a different cell type or revertto a less differentiated cell type. “De-differentiation” refers to theprocess by which a cell reverts to a less specialized (or committed)position within the lineage of a cell. As used herein, the lineage of acell defines the heredity of the cell, i.e., which cells it came fromand what cells it can give rise to. The lineage of a cell places thecell within a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker as compared to anundifferentiated cell. The detectable level of the marker nucleic acidor polypeptide is sufficiently higher or lower in the cells of interestcompared to other cells, such that the cell of interest can beidentified and distinguished from other cells using any of a variety ofmethods known in the art.

As used herein, a cell is “positive for” a specific marker or “positive”when the specific marker is detected in the cell. Similarly, the cell is“negative for” a specific marker, or “negative” when the specific markeris not detected in the cell.

As used herein, “Cell density” and “Seeding Density” are usedinterchangeably herein and refer to the number of cells seeded per unitarea of a solid or semisolid planar or curved substrate.

As used herein, “stage 1” and “S1” are used interchangeably to identifycells expressing markers characteristic of the definitive endoderm (DE).

“Definitive endoderm”, as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express at least one of the following markers:HNF3 beta, GATA4, SOX17, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99,and MIXL1.

“Gut tube”, as used herein, refers to cells derived from definitiveendoderm that express at least one of the following markers: HNF3-beta,HNF1-beta, or HNF4-alpha. Gut tube cells can give rise to all endodermalorgans, such as lungs, liver, pancreas, stomach, and intestine.

Used herein interchangeably are “stage 2” and “S2” which identify cellsexpressing markers characteristic of the primitive gut tube.

“Foregut endoderm” refers to endoderm cells that give rise to esophagus,lungs, stomach, liver, pancreas, gall bladder, and a portion of theduodenum.

“Posterior foregut” refers to endoderm cells that can give rise toposterior stomach, pancreas, liver, and a portion of the duodenum.

“Mid-gut endoderm” refers to endoderm cells that can give rise to theintestines, portions of the duodenum, appendix, and ascending colon.

“Hind-gut endoderm” refers to endoderm cells that can give rise to thedistal third of the transverse colon, the descending colon, sigmoidcolon and rectum.

Both “stage 3” and “S3” are used interchangeably to identify cellsexpressing markers characteristic of the foregut endoderm. “Cellsexpressing markers characteristic of the foregut lineage”, as usedherein, refers to cells expressing at least one of the followingmarkers: PDX1, FOXA2, CDX2, SOX2, and HNF4 alpha.

Used interchangeably herein are “stage 4” and “S4” to identify cellsexpressing markers characteristic of the pancreatic foregut precursor.“Cells expressing markers characteristic of the pancreatic foregutprecursor lineage”, as used herein, refers to cells expressing at leastone of the following markers: PDX1, NKX6.1, HNF6, FOXA2, PTF1a, Prox1and HNF4 alpha.

As used herein, “stage 5” and “S5” are used interchangeably to identifycells expressing markers characteristic of the pancreatic endoderm andpancreatic endocrine precursor cells. “Cells expressing markerscharacteristic of the pancreatic endoderm lineage”, as used herein,refers to cells expressing at least one of the following markers: PDX1,NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4 alpha, SOX9, HB9 or PROX1.Cells expressing markers characteristic of the pancreatic endodermlineage do not substantially express CDX2 or SOX2.

“Pancreatic endocrine cell”, or “Pancreatic hormone expressing cell”, or“Cells expressing markers characteristic of the pancreatic endocrinelineage”, or “Stage 6 cells”, or “S6 cells” are used interchangeablyherein, and refer to a cell capable of expressing at least one of thefollowing hormones: insulin, glucagon, somatostatin, ghrelin, andpancreatic polypeptide.

“Pancreatic insulin positive cell” refers to an endocrine population ofcells expressing insulin, HB9, NKX2.2 and NKX6.1.

“Pancreatic endocrine precursor cell” or “Pancreatic endocrineprogenitor cell” refers to pancreatic endoderm cells capable of becominga pancreatic hormone expressing cell. Such a cell can express at leastone of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6,or ARX.

Used interchangeably herein are “dl”, “d 1”, and “day 1”; “d2”, “d 2”,and “day 2”; “d3”, “d 3”, and “day 3”, and so on. These number lettercombinations refer to a specific day of incubation in the differentstages during the stepwise differentiation protocol of the instantapplication.

“Glucose” and “D-Glucose” are used interchangeably herein and refer todextrose, a sugar commonly found in nature.

Used interchangeably herein are “NeuroD” and “NeuroD 1” which identify aprotein expressed in pancreatic endocrine progenitor cells and the geneencoding it.

Used interchangeably herein are “LDN” and “LDN-193189” to indicate a BMPreceptor inhibitor available from Stemgent, CA, USA.

Isolation, Expansion and Culture of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al. 1998,Science 282:1145-1147). Differentiation of pluripotent stem cells invitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression.Undifferentiated pluripotent stem cells typically have alkalinephosphatase activity, which can be detected by fixing the cells with 4%paraformaldehyde, and then developing with Vector Red as a substrate, asdescribed by the manufacturer (Vector Laboratories, CA, USA).Undifferentiated pluripotent stem cells also typically express OCT4 andTERT, as detected by RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of stem cells canbe confirmed, for example, by injecting cells into SCID mice, fixing theteratomas that form using 4% paraformaldehyde, and then examining themhistologically for evidence of cell types from the three germ layers.Alternatively, pluripotency may be determined by the creation ofembryoid bodies and assessing the embryoid bodies for the presence ofmarkers associated with the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.Pluripotent cells may be readily expanded in culture using variousfeeder layers or by using matrix protein coated vessels. Alternatively,chemically defined surfaces in combination with defined media such asmTesr®1 media (StemCell Technologies, Vancouver, Canada) may be used forroutine expansion of the cells. Pluripotent cells may be readily removedfrom culture plates using enzymatic, mechanical or use of variouscalcium chelators such as EDTA (Ethylenediaminetetraacetic acid).Alternatively, pluripotent cells may be expanded in suspension in theabsence of any matrix proteins or a feeder layer.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily, before approximately 10 to 12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells (hESCs) or human embryonic germ cells, such as, forexample the human embryonic stem cell lines H1, H7, and H9 (WiCellResearch Institute, Madison, Wis., USA). Also suitable are cells takenfrom a pluripotent stem cell population already cultured in the absenceof feeder cells. Also suitable are inducible pluripotent cells (IPS) orreprogrammed pluripotent cells that can be derived from adult somaticcells using forced expression of a number of pluripotent relatedtranscription factors, such as OCT4, NANOG, Sox2, KLF4, and ZFP42 (AnnuRev Genomics Hum Genet 2011, 12:165-185). The human embryonic stem cellsused in the methods of the invention may also be prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science, 1998,282:1145-1147; Curr Top Dev Biol 1998, 38:133-165; Proc Natl Acad SciU.S.A. 1995, 92:7844-7848).

Formation of Cells Expressing Markers Characteristic of the PancreaticEndoderm Lineage from Pluripotent Stem Cells

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, Tra 1-60, and Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6,HNF4 alpha, SOX9, HB9 and PROX1. Suitable for use in the presentinvention is a cell that expresses at least one of the markerscharacteristic of the pancreatic endoderm lineage. In one aspect of thepresent invention, a cell expressing markers characteristic of thepancreatic endoderm lineage is a pancreatic endoderm cell wherein theexpression of PDX1 and NKX6.1 are substantially higher than theexpression of CDX2 and SOX2.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4,ARX, NKX2.2, and PAX6. In one embodiment, a pancreatic endocrine cell iscapable of expressing at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide. Suitable for use inthe present invention is a cell that expresses at least one of themarkers characteristic of the pancreatic endocrine lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the pancreatic endocrine lineage is a pancreaticendocrine cell. The pancreatic endocrine cell may be a pancreatichormone-expressing cell. Alternatively, the pancreatic endocrine cellmay be a pancreatic hormone-secreting cell.

The pancreatic endocrine cells of the invention are cells expressingmarkers characteristic of the β cell lineage. A cell expressing markerscharacteristic of the β cell lineage expresses PDX1 and at least one ofthe following transcription factors: NKX2.2, NKX6.1, NEUROD, ISL1, HNF3beta, MAFA, PAX4, and PAX6. In one aspect of the present invention, acell expressing markers characteristic of the β cell lineage is a βcell.

In an embodiment, the present invention relates to a method of enhancingexpression of insulin and NKX6.1 by culturing a population of stage 5cells in medium comprising Ephrin A4 or Ephrin A3. In some embodiments,the expression of insulin and NKX6.1 is enhanced in the population ofcells to at least 2 times as much as the expression of insulin andNKX6.1 in a population of non-treated cells. In some embodiments, thepopulation of stage 5 cells do not substantially express CDX2 or SOX2.In some embodiments, the population stage 5 cells are obtained by astepwise differentiation of pluripotent cells. In some embodiments, thepluripotent cells are human embryonic pluripotent cells.

In an embodiment, the invention concerns a method of enhancingexpression of somatostatin while suppressing the expression of insulin,glucagon, and ghrelin by culturing stage 5 cells in medium comprisingActivin A or Activin C. In some embodiments, the treated population ofcells expresses at least two times as much somatostatin as non-treatedcultures. In some embodiments, the expression of insulin is suppressedto about half as much as the expression of insulin in non-treatedcultures. In some embodiments, the expression of glucagon is suppressedto about 1/10 as much as the expression of glucagon in non-treatedcultures. In some embodiments, the expression of ghrelin is suppressedto about ⅓ as much as the expression of ghrelin as in non-treatedcultures. In some embodiments, the stage 5 cells do not substantiallyexpress CDX2 or SOX2. In some embodiments, the stage 5 cells areobtained by a stepwise differentiation of pluripotent cells. In someembodiments, the pluripotent cells are human embryonic pluripotentcells.

In an embodiment, the invention refers to a method of enhancingexpression of NKX6.1 by treating stage 5 cells in medium comprisingsemaphorin 3a or Epigen. In some embodiments, the treated population ofcells expresses at least two times as much NKX6.1 as non-treatedcultures. In some embodiments, the level of expression of hormones isnot affected in treated cultures as compared to untreated cultures. Insome embodiments, the stage 5 cells do not substantially express CDX2 orSOX2. In some embodiments, the stage 5 cells are obtained by a stepwisedifferentiation of pluripotent cells. In some embodiments, thepluripotent cells are human embryonic pluripotent cells.

In some embodiments, the present invention relates to a stepwise methodof differentiating pluripotent cells comprising culturing stage 5 cellsin medium comprising Ephrin A4, Ephrin A3, Activin A, Activin C,semaphorin 3a, or Epigen. In some embodiments, the stage 5 cells arecultured in medium comprising Ephrin A4 or Ephrin A3. In someembodiments, the stage 5 cells are cultured in medium comprising ActivinA or Activin C. In some embodiments, the stage 5 cells are cultured inmedium comprising semaphorin 3a, or Epigen. In some embodiments, thepluripotent stem cells are human embryonic pluripotent stem cells.

In an embodiment, the invention relates to a method of inducing insulinexpression comprising culturing pancreatic endoderm cells with an Ephrinligand. In some embodiments, the Ephrin ligand is selected from EphrinA3 and Ephrin A4. In some embodiments, culturing the pancreatic endodermcells with an Ephrin ligand enhances expression of insulin and NKX6.1.In some embodiments, culturing the pancreatic endoderm cells with anEphrin ligand enhances expression of insulin and NKX6.1 in thepancreatic endoderm cells to at least 2 times as much as the expressionof insulin and NKX6.1 in non-treated pancreatic endoderm cells. In someembodiments, the pancreatic endoderm cells do not substantially expressCDX2 or SOX2. In some embodiments, the pancreatic endoderm cells areobtained by a stepwise differentiation of pluripotent stem cells. Insome embodiments, the pluripotent stem cells used in the methods of theinvention are human embryonic pluripotent stem cells.

In an embodiment, the invention concerns insulin and NKX6.1-expressingcells prepared by the methods of the invention.

In an embodiment, the invention refers to a method for inducingendocrine cluster formation comprising culturing pancreatic endodermcells with a sphingosine-1 receptor agonist. In some embodiments, thepancreatic endoderm cells are obtained by a stepwise differentiation ofpluripotent stem cells. In some embodiments, the pluripotent stem cellsare human embryonic pluripotent stem cells.

Publications cited throughout this document are hereby incorporated byreference in their entirety. The present invention is furtherillustrated, but not limited, by the following examples.

EXAMPLES Example 1 Identification of EphrinA4 as a Strong Inducer ofInsulin Expression

This example was carried out to understand the role of various proteinson the generation of pancreatic endoderm/endocrine cultures from thedifferentiation of human ES cells.

Cells of the human embryonic stem cell line H1 (hESC H1, passage 40)were seeded as single cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30dilution; BD Biosciences, NJ)-coated dishes in mTeSR®1 media (StemCellTechnologies, Vancouver, Canada) supplemented with 10 μM of Y27632 (Rockinhibitor, Catalog No. Y0503, SigmaAldrich, St. Louis, Mo.). Forty-eighthours post seeding, cultures were washed in incomplete PBS (phosphatebuffered saline without Mg or Ca). Cultures were differentiated intopancreatic endoderm/endocrine lineages as follows:

-   a) Stage 1 (Definitive Endoderm (DE)—3 days): Cells were cultured    for one day in stage 1 media: MCDB-131 medium (Catalog No.    10372-019, Invitrogen, Carlsbad, Calif.) supplemented with 0.1%    fatty acid-free BSA (Catalog No. 68700, Proliant, Ankeny, Iowa),    0.0012 g/ml sodium bicarbonate (Catalog No. 53187, SigmaAldrich, St.    Louis, Mo.), 1× GlutaMax™ (Invitrogen Catalog No. 35050-079), 4.5 mM    D-Glucose (SigmaAldrich Catalog No. G8769), 100 ng/ml GDF8 (R&D    Systems, Minneapolis, Minn.) and 1 μM MCX compound (a GSK3B    inhibitor, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo    [19.3.1.1˜2,6˜.1˜8,12˜]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one,    US Patent Application Publication No. 2010-0015711; incorporated    herein by reference in its entirety). Cells were then cultured for    additional day in MCDB-131 medium supplemented with 0.1% fatty    acid-free BSA, 0.0012 g/ml sodium bicarbonate, 1× GlutaMax™, 4.5 mM    D-Glucose, 100 ng/ml GDF8, and 0.1 μM MCX compound. Cells were then    cultured for an additional day in MCDB-131 medium supplemented with    0.1% fatty acid-free BSA, 0.0012 g/ml sodium bicarbonate, 1×    GlutaMax™, 4.5 mM D-Glucose, and 100 ng/ml GDF8, then-   b) Stage 2 (Primitive gut tube—2 days): Cells were treated for two    days with MCDB-131 medium supplemented with 0.1% fatty acid-free    BSA; 0.0012 g/ml sodium bicarbonate; 1× GlutaMax™; 4.5 mM D-Glucose;    0.25 mM ascorbic acid (Sigma, St. Louis, Mo.) and 25 ng/ml FGF7 (R &    D Systems, Minneapolis, Minn.), then-   c) Stage 3 (Foregut-2 days): Cells were treated with MCDB-131 medium    supplemented with a 1:200 dilution of ITS-X (Invitrogen); 4.5 mM    Glucose; 1× GlutaMax™; 0.0017 g/ml sodium bicarbonate; 2% fatty    acid-free BSA; 0.25 μM SANT-1 (Sigma, St. Louis, Mo.); 10 ng/ml of    Activin-A (R & D Systems); 1 μM retinoic acid (RA; Sigma); 25 ng/ml    FGF 7; 0.25 mM ascorbic acid; 200 nM TPB (a PKC activator; Catalog    No. 565740; EMD Chemicals, Gibstown, N.J.); 10 μM forskolin (FSK,    Sigma), and 100 nM LDN (a BMP receptor inhibitor; Catalog No.    04-0019; Stemgent; San Diego, Calif.) for day 1. On day 2, cells    were treated with MCDB-131 medium supplemented with a 1:200 dilution    of ITS-X; 4.5 mM Glucose; 1× GlutaMax™; 0.0017 g/ml sodium    bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 10 ng/ml of    Activin A; 1 μM RA; 25 ng/ml FGF7; 0.25 mM ascorbic acid, 200 nM    TPB, 10 μM forskolin and 10 nM LDN, then-   d) Stage 4 (Pancreatic foregut precursor—2 days); Cells were treated    with MCDB-131 medium supplemented with a 1:200 dilution of ITS-X;    4.5 mM Glucose; 1× GlutaMax™; 0.0015 g/ml sodium bicarbonate; 2%    fatty acid-free BSA; 0.25 μM SANT-1; 50 nM RA; 50 nM LDN-193189; 10    μM forskolin; 0.25 mM ascorbic acid; and 100 nM TPB for two days,    then-   e) Stage 5 (Pancreatic endoderm/endocrine—3 days): Stage 4 cells    were treated with MCDB-131 medium supplemented with a 1:200 dilution    of ITS-X; 20 mM Glucose; 1× GlutaMax™; 0.0015 g/ml sodium    bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50 nM RA; 10 μM    forskolin; 0.25 mM ascorbic acid for three days, with the addition    of 100 nM ALk5 inhibitor SD-208 (disclosed in Molecular Pharmacology    2007, 72:152-161) for days 2-3 only.

At day 1 of stage 5, the factors listed in Table I, below, were spikedinto the media and upon completion of S5 (day 3 of stage 5) mRNA wascollected for PCR analysis of relevant pancreatic endoderm/endocrinegenes. As a control, cultures were treated only with the S5 media listedabove. Total RNA was extracted with the RNeasy Mini Kit (Qiagen;Valencia, Calif.) and reverse-transcribed using a High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. cDNA was amplified usingTaqman Universal Master Mix and Taqman Gene Expression Assays which werepre-loaded onto custom Taqman Arrays (Applied Biosystems). Data wereanalyzed using Sequence Detection Software (Applied Biosystems) andnormalized to undifferentiated human embryonic stem (hES) cells usingthe AACt method. All primers were purchased from Applied Biosystems.

TABLE I List of factors tested at S5 of Example 1 Protein ConcentrationR & D Systems Catalogue Number Epigen 20 ng/ml 6629-EP-025 Semaphorin 3a50 ng/ml 1250-S3-025 Netrin 4 100 ng/ml  1254-N4-025 Galectin-8 100ng/ml  1305-GA-050 Tryptase-Y-1 20 ng/ml 1667-SE-010 BetaCellulin 20ng/ml 261-CE-010 Lumican 100 ng/ml  2846-LU-050 Epimorphin 50 ng/ml2936-EP-025 Mesothelin 50 ng/ml 3265-MS-050 Matrilin-4 100 ng/ml 3380-MN-050 Meteorin 50 ng/ml 3475-MN-025 Ephrin-A4 100 ng/ml  369-EAIBSP 100 ng/ml  4014-SP-050 EFG-L6 50 ng/ml 4329-EG-025 R-Spondin-1 100ng/ml  4645-RS-025 Ephrin-B1 100 ng/ml  473-EB-200 Hepsin 50 ng/ml4776-SE-010 Activin A 20 ng/ml 338-AC-010 EphA4 50 ng/ml 6827-A4-050Neurocan 100 ng/ml  6508-NC-050 DKK1 100 ng/ml  5439-DK-010 Kallikrein-450 ng/ml 1719-SE-010 EGF 20 ng/ml 236-EG-200 BDNF 20 ng/ml 248-BD-005Spinesin 50 ng/ml 2495-SE-010 HGF 20 ng/ml 294-HG-005 EphB4 50 ng/ml3038-B4-100 Relaxin1 50 ng/ml 3257-RN-025 Activin C 20 ng/ml 4879-AC-010BMP5 20 ng/ml 615-BMC-020 IGF-1 20 ng/ml 291-G1-200

FIG. 1A to FIG. 1G depict data from real-time PCR analyses of theexpression of the following genes in cells of the human embryonic stemcell line H1 differentiated to stage 5 as outlined in Example 1 and inthe presence of factors listed in Table I: Insulin (FIG. 1A),somatostatin (FIG. 1B), ghrelin (FIG. 1C), glucagon (FIG. 1D), PDX1(FIG. 1E), NKX6.1 (FIG. 1F), and NGN3 (FIG. 1G).

As shown in FIG. 1, Ephrin-A4 enhanced mRNA expression of NKX6.1 andinsulin as compared to control cultures (FIG. 1F) while showing minimalimpact on PDX1 (FIG. 1E) and NGN3 expression (FIG. 1G). Factors such asActivin-A and Activin-C significantly enhanced expression ofsomatostatin (FIG. 1B) while suppressing the expression of insulin (FIG.1A), glucagon (FIG. 1D), and ghrelin (FIG. 1C). Moreover, factors suchas semaphorin 3a and Epigen enhanced NKX6.1 expression while notaffecting expression of hormones as compared to untreated cultures. InFIG. 1A to FIG. 1G, the average level of expression of the differentmarkers in control cultures are shown by a dotted line on the graphs.

Example 2 Verification of the Effect of Ephrins on Insulin Expression atS5

This example describes the validation of hits identified in Example 1.In particular, the effect of addition of Ephrin-A3 or Ephrin-A4 at S5 inthe protocol listed below.

Cells of the human embryonic stem cell line H1 (hESC H1, passage 40)were seeded as single cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30dilution; BD Biosciences, NJ)-coated dishes in mTeSR®1 mediasupplemented with 10 μM of Y27632. Forty-eight hours post seeding,cultures were washed in incomplete PBS (phosphate buffered salinewithout Mg or Ca). Cultures were differentiated into pancreaticendoderm/endocrine lineages as follows:

-   -   a) Stage 1 (Definitive Endoderm (DE)—3 days): Cells were        cultured for one day in stage 1 media (see Example 1, above).        Cells were then cultured for an additional day in MCDB-131        medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g/ml        sodium bicarbonate, 1× GlutaMax™, 4.5 mM D-Glucose, 100 ng/ml        GDF8, and 0.1 μM MCX compound. Cells were then cultured for an        additional day in MCDB-131 medium supplemented with 0.1% fatty        acid-free BSA, 0.0012 g/ml sodium bicarbonate, 1× GlutaMax™, 4.5        mM D-Glucose, and 100 ng/ml GDF8, then    -   b) Stage 2 (Primitive gut tube—2 days): Cells were treated for        two days with MCDB-131 medium supplemented with 0.1% fatty        acid-free BSA; 0.0012 g/ml sodium bicarbonate; 1× GlutaMax™; 4.5        mM D-Glucose; 0.25 mM ascorbic acid (Sigma, MO) and 25 ng/ml        FGF7 (R & D Systems, MN), then    -   c) Stage 3 (Foregut—2 days): Cells were treated with MCDB-131        medium supplemented with a 1:200 dilution of ITS-X (Invitrogen,        Ca); 4.5 mM Glucose; 1× GlutaMax™; 0.0017 g/ml sodium        bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1 (Sigma, MO);        10 ng/ml of Activin-A (R& D Systems, MN); 1 μM RA (Sigma, MO);        25 ng/ml FGF7; 0.25 mM ascorbic acid; 200 nM TPB (PKC activator;        Catalog No. 565740; EMD Chemicals, Gibstown, N.J.); 10 μM        forskolin and 100 nM LDN (BMP receptor inhibitor; Catalog No.        04-0019; Stemgent) for day 1. On day 2, cells were treated with        MCDB-131 medium supplemented with a 1:200 dilution of ITS-X; 4.5        mM Glucose; 1× GlutaMax™; 0.0017 g/ml sodium bicarbonate; 2%        fatty acid-free BSA; 0.25 μM SANT-1; 10 ng/ml of Activin-A; 1 μM        RA; 25 ng/ml FGF7; 0.25 mM ascorbic acid, 200 nM TPB, 10 μM        forskolin and 10 nM LDN, then    -   d) Stage 4 (Pancreatic foregut precursor—2 days): Cells were        treated with MCDB-131 medium supplemented with a 1:200 dilution        of ITS-X; 4.5 mM Glucose; 1× GlutaMax™; 0.0015 g/ml sodium        bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50 nM RA;        50 nM LDN-193189; 10 μM forskolin; 0.25 mM ascorbic acid; and        100 nM TPB for two days, then    -   e) Stage 5 (Pancreatic endoderm/endocrine—3 days): Stage 4 cells        were treated with MCDB-131 medium supplemented with a 1:200        dilution of ITS-X; 4.5 mM Glucose; 1× GlutaMax™; 0.0015 g/ml        sodium bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50        nM RA; 10 μM forskolin; 0.25 mM ascorbic acid; 100 nM ALk5        inhibitor (for days 2-3 only) (SD-208, disclosed in Molecular        Pharmacology 2007, 72:152-161) and +/−0-100 ng/ml of Ephrin-A3        or Ephrin-A4 (R & D systems, MN) for three days.

At the end of Stage 5, control and Ephrin-treated cultures were fixedand stained for insulin protein expression (using Guinea Piganti-insulin antibody from Millipore; Cambridge, Mass.). FIG. 2A to FIG.2C depic images of cells immunostained for insulin. FIG. 2A, controlcells; FIG. 2B, cells treated with 50 ng/ml Ephrin A3; FIG. 2C cellstreated with 100 ng/ml Ephrin A3. FIG. 3A to FIG. 3C depicts images ofcells immunostained for insulin. FIG. 3A control cells; FIG. 3B, cellstreated with 50 ng/ml Ephrin A4; FIG. 3C cells treated with 100 ng/mlEphrin A4. These data show that, consistent with data from Example 1,addition of both Ephrin-A3 and Ephrin-A4 at stage 5 significantlyenhanced protein expression of insulin.

Example 3 Addition of Sphingoisne-1-Phosphate at S6 SignificantlyAccelerates Formation of Cell Clusters Containing Endocrine Hormones

This example describes the progression of endocrine cluster formation atstage 6 and the effect of sphingosine-1-phosphate in accelerating theformation of the endocrine rich clusters.

Cells of the human embryonic stem cell line H1 (hESC H1, passage 40)were seeded as single cells at 1×10⁵ cells/cm² on MATRIGEL™ (1:30dilution; BD Biosciences, NJ) coated dishes in mTeS®101 media (StemCellTechnologies, Vancouver, Canada) supplemented with 10 μM of Y27632.Forty-eight hours post seeding, cultures were washed in incomplete PBS(phosphate buffered saline without Mg or Ca). Cultures weredifferentiated into pancreatic endoderm/endocrine lineages as follows:

-   -   a) Stage 1 (Definitive Endoderm (DE)—3 days): Cells were        cultured for one day in stage 1 media (see Example 1, above).        Cells were then cultured for an additional day in MCDB-131        medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g/ml        sodium bicarbonate, 1× GlutaMax™, 4.5 mM D-Glucose, 100 ng/ml        GDF8, and 0.1 μM MCX compound. Cells were then cultured for an        additional day in MCDB-131 medium supplemented with 0.1% fatty        acid-free BSA, 0.0012 g/ml sodium bicarbonate, 1× GlutaMax™, 4.5        mM D-Glucose, and 100 ng/ml GDF8, then    -   b) Stage 2 (Primitive gut tube—2 days): Cells were treated for        two days with MCDB-131 medium supplemented with 0.1% fatty        acid-free BSA; 0.0012 g/ml sodium bicarbonate; 1× GlutaMax™; 4.5        mM D-Glucose; 0.25 mM ascorbic acid (Sigma, MO) and 25 ng/ml        FGF7 (R & D Systems, MN), then    -   c) Stage 3 (Foregut—2 days): Cells were treated with MCDB-131        medium supplemented with a 1:200 dilution of ITS-X (Invitrogen,        Ca); 4.5 mM Glucose; 1× GlutaMax™; 0.0017 g/ml sodium        bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1 (Sigma, MO);        10 ng/ml of Activin-A (R& D Systems, MN); 1 μM RA (Sigma, MO);        25 ng/ml FGF7; 0.25 mM ascorbic acid; 200 nM TPB (PKC activator;        Catalog No. 565740; EMD Chemicals, Gibstown, N.J.); 10 μM        forskolin (FSK, Sigma, MO), and 100 nM LDN (BMP receptor        inhibitor; Catalog No. 04-0019; Stemgent, CA) for day 1. On day        2, cells were treated with MCDB-131 medium supplemented with a        1:200 dilution of ITS-X; 4.5 mM Glucose; 1× GlutaMax™; 0.0017        g/ml sodium bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1;        10 ng/ml of Activin-A; 1 μM RA; 25 ng/ml FGF7; 0.25 mM ascorbic        acid, 200 nM TPB, and 10 nM LDN, then    -   d) Stage 4 (Pancreatic foregut precursor—2 days); Cells were        treated with MCDB-131 medium supplemented with a 1:200 dilution        of ITS-X; 4.5 mM Glucose; 1× GlutaMax™; 0.0015 g/ml sodium        bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50 nM RA;        50 nM LDN-193189; 10 μM forskolin; 0.25 mM ascorbic acid; 2        ng/ml FGF7; 1 ng/ml AA; and 100 nM TPB for two days, then    -   e) Stage 5 (Pancreatic endoderm/endocrine—3 days): Stage 4 cells        were treated with MCDB-131 medium supplemented with a 1:200        dilution of ITS-X; 15 mM Glucose; 1× GlutaMax™; 0.0015 g/ml        sodium bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50        nM RA; 10 μM forskolin; 0.25 mM ascorbic acid; and 1 ng/ml FGF7        for three days; with the addition of 100 nM ALKS inhibitor        SD-208 at days 2-3 only, then    -   f) Stage 6 (Pancreatic endocrine—3-10 days): Stage 5 cells were        treated with MCDB-131 medium supplemented with a 1:200 dilution        of ITS-X; 15 mM Glucose; 1× GlutaMax™; 0.0015 g/ml sodium        bicarbonate; 2% fatty acid-free BSA; 0.25 μM SANT-1; 50 nM RA;        0.25 mM ascorbic acid; for 3-10 days. In some cultures 10 μM of        Sphingosine-1-phosphate (Sigma, MO) was added for three days.

FIG. 4A to FIG. 4D depict phase contrast images of S6 cultures of cellstreated with sphingosine-1-phosphate (S1P) and imaged on day 1 (FIG.4A), day 7 (FIG. 4B), and at two different magnifications at day 10(FIG. 4C and FIG. 4D). The images show that on day 7, there was clearevidence of clustering of endocrine cells and on day 10 the clusterswere separated from each other by a thin layer of pancreatic endodermepithelium.

FIG. 5A to FIG. 5D depict images of cells immunostained for Hb9 (FIG.5A) and NKX6.1 (FIG. 5B), or immunostained for insulin (FIG. 5C) and Hb9(FIG. 5D). FIG. 5A and FIG. 5B show that the endocrine clusters wereenriched for Hb9 while the pancreatic epithelium surrounding theclusters were enriched for NKX6.1. Some of the cells in the Hb9-enrichedclusters were also positive for NKX6.1. The clusters were enriched forinsulin and Hb9 as shown in FIG. 5C and FIG. 5D. This morphologicalchange closely resembles pancreatic development where NKX6.1+PDX1+richepithelium gives rise to endocrine clusters. In each instance, the pairof images was obtained using different filters from the same field ofcells.

FIG. 6A and FIG. 6B depict phase contrast images, at differentmagnifications, of cells treated with 10 μM sphingosine-1-phosphate(S1P) and harvested three days after start of stage 6. These images showthat endocrine clusters emerged only 3 days after start of stage 6. Thisis about 7 days earlier than formation of the clusters in controlcultures.

FIG. 6C and FIG. 6D depict images of control cells (FIG. 6C) and cellstreated with S1P (FIG. 6D) immunostained for NKX2.2. In S1P-treatedcultures, the endocrine clusters were also enriched for NKX2.2+ cells(FIG. 6C), as compared to control cultures where NKX2.2+ cells weredistributed uniformly across the culture (FIG. 6D).

What is claimed is:
 1. A method for inducing formation of endocrine clusters, comprising culturing pancreatic endocrine cells with a sphingosine-1 receptor agonist.
 2. The method of claim 1, wherein the sphingosine-1 receptor agonist is sphingosine-1-phosphate (S1P).
 3. The method of claim 1, wherein the pancreatic endocrine cells are obtained by a stepwise differentiation of pluripotent stem cells.
 4. The method of claim 3, wherein the pluripotent stem cells are human embryonic pluripotent stem cells.
 5. The method of claim 3, wherein the pluripotent stem cells are human pluripotent stem cells.
 6. The method of claim 1, wherein the method further comprises: differentiating pluripotent stem cells into definitive endoderm cells; differentiating the definitive endoderm cells into primitive gut tube cells; differentiating the primitive gut tube cells into foregut cells; differentiating the foregut cells into pancreatic foregut precursor cells; differentiating the foregut precursor cells into the pancreatic endoderm; and differentiating the pancreatic endoderm cells into pancreatic endocrine cells.
 7. The method of claim 1, wherein the pancreatic endocrine cells are human pancreatic endocrine cells. 